4-[3,5-bis-(2-hydroxy-phenyl)-[1,2,4]triazol-1-yl]-benzoic acid derivatives for treating an excess of metal in the body

ABSTRACT

The present invention provides new 4-[3,5-bis-(2-hydroxy-phenyl)-[1,2,4]triazol-1-yl]-benzoic acid derivatives which can be used in the treatment of diseases which cause an excess of metal in the human or animal body or are caused by it.

Various disorders of warm-blooded animals are linked with an excess ofmetals, in particular trivalent metals, in the body tissues. For examplealuminium in dialysis encephalopathy and osteomalacia, as well as inAlzheimer's disease. In other illnesses, in particular of man, an excessof iron occurs in the various tissues. This is designated as ironoverload (formerly haemosiderosis). It occurs, for example, afterparenteral administration of iron (especially repeated bloodtransfusions) or after increased uptake of iron from thegastrointestinal tract. Repeated transfusions are necessary in seriousanaemias, especially in thalassaemia major, the severe form ofβ-thalassaemia, but also in other anaemias. Increased iron absorptionfrom the gastrointestinal tract either takes place primarily, e.g. onaccount of a genetic defect (so-called haemochromatosis), orsecondarily, such as after anaemias in which blood transfusions are notnecessary, for example thalassaemia intermedia, a milder form ofβ-thalassaemia.

Untreated iron overload can cause severe organ damage, in particular ofthe liver, the heart and the endocrine organs, and can lead to death.Iron chelators are able to mobilize and excrete the iron deposited inthe organs and thus lower the iron-related morbidity and mortality.

A reduction in the iron(III) concentration is also of interest for thetreatment of disorders due to iron(III)-dependent microorganisms andparasites, which is of key importance not only in human medicine, suchas in particular in malaria, but also in veterinary medicine. Complexingof other metals, in partcular trivalent metals, can also be used forexcretion thereof from the organism. A number of further applicationsare also described in the literature, e.g. by G. Kontoghiorghes,Toxicology Lett. 80, 1-18 (1995).

Desferrioxamine B has already been known for a long time and usedtherapeutically for these purposes (H. Bickel, H. Keberle and E.Vischer, Helv. Chim. Acta 46, 1385-9 [1963]). A disadvantage of thispreparation, however, turns out to be the fact that desferrioxamine andits salts only have a low, inadequate activity on oral administrationand require a parenteral administration form in all of theabovementioned application possibilities. It is thus recommended, forexample, as a particularly effective method to administer the activesubstance by means of a slow (8- to 12-hour) subcutaneous infusion,which, however, demands the use of a portable mechanical device, such asan infusion syringe actuated by an electrical drive. Apart from theirawkwardness, such solutions are affected by a high treatment cost, whichseverely restricts their use; in particular a comprehensive treatment ofthe thalassaemias in the countries of the Mediterranean region, of theMiddle East, India and South-East Asia, of malaria worldwide and ofsickle-cell anaemia in African countries is made impossible. Thesewidespread diseases are furthermore a serious problem for the healthservice in these countries and make the search for a simpler and moreinexpensive therapy, preferably by means of an orally activepreparation, the urgent object in this area.

The present invention provides new4-[3,5-bis-(2-hydroxy-phenyl)-[1,2,4]triazol-1-yl]-benzoic acidderivatives which can be used in the treatment of diseases which causean excess of metal in the human or animal body or are caused by it.

The present invention relates to compounds of the formula I

wherein one of the radicals R₁, R₂, R₃ and R₄ is hydroxy and theremaining radicals are each independently of the others hydrogen orhydroxy;and salts thereof.

Salts are especially the pharmaceutically acceptable salts of compoundsof formula I.

Such salts are formed, for example, as acid addition salts, preferablywith organic or inorganic acids, from compounds of formula I with abasic nitrogen atom, especially the pharmaceutically acceptable salts.

In the presence of negatively charged radicals, such as carboxy orsulfo, salts may also be formed with bases, e.g. metal or ammoniumsalts, such as alkali metal or alkaline earth metal salts, or ammoniumsalts with ammonia or suitable organic amines, such as tertiarymonoamines.

In the presence of a basic group and an acid group in the same molecule,a compound of formula I may also form internal salts.

For isolation or purification purposes it is also possible to usepharmaceutically unacceptable salts, for example picrates orperchlorates. Only the pharmaceutically acceptable salts or freecompounds (if the occasion arises, in the form of pharmaceuticalcompositions) attain therapeutic use, and these are therefore preferred.

In view of the close relationship between the novel compounds in freeform and in the form of their salts, including those salts that can beused as intermediates, for example in the purification or identificationof the novel compounds, hereinbefore and hereinafter any reference tothe free compounds is to be understood as referring also to thecorresponding salts, as appropriate and expedient.

The compounds of formula I have valuable pharmacological properties whenused in the treatment of disorders which cause an excess of metal in thehuman or animal body or are caused by it, primarily a marked binding oftrivalent metal ions, in particular those of iron (A. E. Martell and R.J. Motekaitis, “Determination and Use of Stability Constants”, VCHPublishers, New York 1992). They are able, for example in an animalmodel using the non-iron overloaded cholodocostomized rat (R. J.Bergeron et al., J. Med. Chem. 34, 2072-2078 (1991)) or theiron-overloaded monkey (R. J. Bergeron et al., Blood 81, 2166-2173(1993)) in doses from approximately 5 μmol/kg, inter alia, to preventthe deposition of iron-containing pigments and in the case of existingiron deposits in the body cause excretion of the iron.

Very special preference is given to a compound of the formula I which isselected from the group consisting of4-[3-(2,3-dihydroxy-phenyl)-5-(2-hydroxy-phenyl)-[1,2,4]triazol-1-yl]-benzoicacid,[3-(2,5-dihydroxy-phenyl)-5-(2-hydroxy-phenyl)-[1,2,4]triazol-1-yl]-benzoicacid,4-[5-(2,5-dihydroxy-phenyl)-3-(2-hydroxy-phenyl)-[1,2,4]triazol-1-yl]-benzoicacid, and salts thereof.

A compound of the invention may be prepared by processes known per sefor other compounds. In particular, they can be prepared by theprocesses described in e.g. WO 97/49395.

In the preferred embodiment, a compound of the formula I is preparedaccording to the processes and process steps defined in the Examplesbelow.

Pharmaceutical Compositions, Methods, and Uses

In particular, the invention relates to the use of a compound of formulaI for the treatment of diseases which cause an excess of iron in thehuman or animal body or are caused by it, preferably in the form ofpharmaceutically acceptable preparations, in particular in a method forthe therapeutic treatment of the human body, and to a treatment methodof this type.

In addition, the invention relates to novel preparations, comprising atleast one compound of the formula I and salts thereof; and at least onepharmaceutically acceptable carrier; and to methods for theirpreparation. These pharmaceutical preparations are those for enteral, inparticular oral, and furthermore rectal, administration and those forparenteral administration to warm-blooded animals, especially to man,the pharmacological active ingredient being contained on its own ortogether with customary pharmaceutical adjuncts. The pharmaceuticalpreparations contain (in percentages by weight), for example, fromapproximately 0.001% to 100%, preferably from approximately 0.1% toapproximately 100%, of the active ingredient.

Pharmaceutical preparations for enteral or parenteral administrationare, for example, those in unit dose forms, such as sugar-coatedtablets, tablets, dispersible tablets, effervescent tablets, capsules,suspendable powders, suspensions or suppositories, or ampoules. Theseare prepared in a manner known per se, e.g. by means of conventionalpan-coating, mixing, granulation or lyophilization processes.Pharmaceutical preparations for oral administration can thus be obtainedby combining the active ingredient with solid carriers, if desiredgranulating a mixture obtained and processing the mixture or granules,if desired or necessary, after addition of suitable adjuncts to givetablets or sugar-coated tablet cores.

Suitable carriers are, in particular, fillers such as sugars, e.g.lactose, sucrose, mannitol or sorbitol, cellulose preparations and/orcalcium phosphates, e.g. tricalcium phosphate or calcium hydrogenphosphate, furthermore binders, such as starch pastes, using, forexample, maize, wheat, rice or potato starch, gelatin, tragacanth,methylcellulose and/or polyvinylpyrrolidone, and, if desired,disintegrants, such as the abovementioned starches, furthermorecarboxymethyl starch, crosslinked polyvinylpyrrolidone, agar or alginicacid or a salt thereof, such as sodium alginate. Adjuncts are primarilyflow-regulating and lubricating agents, e.g. salicylic acid, talc,stearic acid or salts thereof, such as magnesium or calcium stearate,and/or polyethylene glycol. Sugar-coated tablet cores are provided withsuitable, if desired enteric, coatings, using, inter alia, concentratedsugar solutions which, if desired, contain gum arabic, talc,polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide,coating solutions in suitable organic solvents or solvent mixtures or,for the preparation of enteric coatings, solutions of suitable cellulosepreparations, such as acetylcellulose phthalate orhydroxypropylmethylcellulose phthalate. Colorants or pigments, e.g. forthe identification or the marking of various doses of active ingredient,can be added to the tablets or sugar-coated tablet coatings.

Dispersible tablets are tablets which rapidly disintegrate in acomparatively small amount of liquid, e.g. water, and which, if desired,contain flavourings or substances for masking the taste of the activeingredient. They can advantageously be employed for the oraladministration of large individual doses, in which the amount of activeingredient to be administered is so large that on administration as atablet which is to be swallowed in undivided form or without chewingthat it can no longer be conveniently ingested, in particular bychildren. Further orally administrable pharmaceutical preparations arehard gelatin capsules and also soft, closed capsules of gelatin and aplasticizer, such as glycerol or sorbitol. The hard gelatin capsules cancontain the active ingredient in the form of granules, e.g. as a mixturewith fillers, such as lactose, binders, such as starches, and/orglidants, such as talc or magnesium stearate, and, if desired,stabilizers. In soft capsules, the active ingredient is preferablydissolved or suspended in suitable liquids, such as fatty oils, liquidparaffin or liquid polyethylene glycols, it also being possible to addstabilizers.

Moreover, suspendable powders, e.g. those which are described as “powderin bottle”, abbreviated “PIB”, or ready-to-drink suspensions, aresuitable for an oral administration form. For this form, the activeingredient is mixed, for example, with pharmaceutically acceptablesurface-active substances, for example, sodium lauryl sulfate orpolysorbate, suspending auxiliaries, e.g. hydroxypropylcellulose,hydroxypropylmethylcellulose or another known from the prior art andpreviously described, for example, in “Handbook of PharmaceuticalEcipients”, pH regulators, such as citric or tartaric acid and theirsalts or a USP buffer and, if desired, fillers, e.g. lactose, andfurther auxiliaries, and dispensed into suitable vessels, advantageouslysingle-dose bottles or ampoules. Immediately before use, a specificamount of water is added and the suspension is prepared by shaking.Alternatively, the water can also be added even before dispensing.

Rectally administrable pharmaceutical preparations are, for example,suppositories which consist of a combination of the active ingredientwith a suppository base. A suitable suppository base is, for example,natural or synthetic triglycerides, paraffin hydrocarbons, polyethyleneglycols or higher alkanols. Gelatin rectal capsules can also be usedwhich contain a combination of the active ingredient with a basesubstance. Possible base substances are, for example, liquidtriglycerides, polyethylene glycols or paraffin hydrocarbons.

For parenteral administration, aqueous solutions of an active ingredientin water-soluble form, e.g. of a water-soluble salt, are primarilysuitable; furthermore suspensions of the active ingredient, such asappropriate oily injection suspensions, suitable lipophilic solvents orvehicles, such as fatty oils, e.g. sesame oil, or synthetic fatty acidesters, e.g. ethyl oleate or triglycerides, being used, or aqueousinjection suspensions which contain viscosity-increasing substances,e.g. sodium carboxymethylcellulose, sorbitol and/or dextran, and, ifdesired, also stabilizers.

The dosage of the active ingredient can depend on various factors, suchas activity and duration of action of the active ingredient, severity ofthe illness to be treated or its symptoms, manner of administration,warm-blooded animal species, sex, age, weight and/or individualcondition of the warm-blooded animal. The doses to be administered dailyin the case of oral administration are between 10 and approximately 120mg/kg, in particular 20 and approximately 80 mg/kg, and for awarm-blooded animal having a body weight of approximately 40 kg,preferably between approximately 400 mg and approximately 4,800 mg, inparticular approximately 800 mg to 3,200 mg, which is expedientlydivided into 2 to 12 individual doses.

Preferably, the invention relates to novel preparations comprising atleast one compound of the formula I and salts thereof; and at least onepharmaceutically acceptable carrier, and to methods for theirpreparation. These pharmaceutical preparations are those for enteral, inparticular oral, and furthermore rectal, administration, and those forparenteral administration to warm-blooded animals, especially to man,the pharmacological active ingredient being present on its own ortogether with customary pharmaceutical adjuncts. The pharmaceuticalpreparations contain (in percentages by weight), for example, fromapproximately 0.001% to 100%, preferably from approximately 0.1% toapproximately 50%, of the active ingredient.

EXAMPLES

The following Examples serve to illustrate the invention withoutlimiting its scope.

Temperatures are measured in degrees Celsius. Unless otherwiseindicated, the reactions take place at room temperature.

The R_(f) values which indicate the ratio of the distance moved by eachsubstance to the distance moved by the eluent front are determined onsilica gel thin-layer plates (Merck, Darmstadt, Germany) by thin-layerchromatography using the respective named solvent systems.

The analytical HPLC conditions are as follows: Instrument: HP-1100System with G1311A quaternery pump (0.8 ml dead volume), G1313Aautosampler, G1316A column compartment (35° C.), G1315A diode arraydetector and G1946A mass spectrometer. Column: Waters Symmetry C8, 50 ×2.1 mm 3.5 μm mean particle size. Detection by UV absorption at 210-250nm. The retention times (t_(R)) are given in minutes. Flow rate: 0.5 ml/min. Gradient: 5% → 95% b) in a) within 6.5 min. a): water + 5%acetonitrite + 0.1% TFA b): Acetonitrile + 0.1% TFA.

The short forms and abbreviations used have the following definitions:

-   h hour(s)-   MS-ES mass spectroscopy (electron spray)-   min minute(s)-   m.p. melting point-   MS-APCI⁺ mass spectroscopy (atomic pressure chemical ionisation)-   t_(R) retention times.

Example 14-[3-(2,3-Dihydroxy-phenyl)-5-(2-hydroxy-phenyl)-[1,2,4]triazol-1-yl]benzoicAcid (M2)

A mixture of 5.5 g of 2-(2,3-dihydroxy-phenyl)benzo[e][1,3]oxazin-4-onewith 8-hydroxy-2-(2-hydroxy-phenyl)-benzo[e][1,3]oxazin-4-one and 3.3 g4-hydrazino-benzoic acid in 50 ml of ethanol is heated to reflux for 2.The mixture is evaporated to dryness and the product is isolated bypreparative HPLC; HPLC t_(R)=5.15 min, MS-APCI⁺: (M+H)⁺=390, m.p.:223-226° C.

Step 1.1: Mixture of 2-(2,3-Dihydroxy-phenyl)-benzo[e][1,3]oxazin-4-onewith 8-Hydroxy-2-(2-hydroxy-phenyl)-benzo[e][1,3]oxazin-4-one

A mixture of 10 g 2,3-dihydroxy-benzoic acid, 7.5 g 2-hydroxy-benzamideand 0.5 ml pyridine in 50 ml toluene is heated under reflux. 8.8 ml ofthionylchloride is added over a period of 1 h. The mixture is kept underreflux for another hour, cooled to 40° C. and 100 ml ethanol are added.After cooling to 5° C. the precipitated product is filtered off andwashed with cold ethanol; HPLC t_(R)=4.63 min, MS-APCI⁺: (M+H)⁺=256.

Example 24-[3-(2,5-Dihydroxy-phenyl)-5-(2-hydroxy-phenyl)-[1,2,4]triazol-1-yl]-benzoicAcid (M1) and4-[5-(2,5-Dihydroxy-phenyl)-3-(2-hydroxy-phenyl)-[1,2,4]triazol-1-yl]-benzoicAcid (M4)

A mixture of 5 g of4-[3-(2-hydroxy-5-methoxy-phenyl)-5-(2-hydroxy-phenyl)-[1,2,4]triazol-1-yl]-benzoicacid with4-[5-(2-hydroxy-5-methoxy-phenyl)-3-(2-hydroxy-phenyl)-[1,2,4]triazol-1-yl]-benzoicacid is dissolved in 40 ml acetic acid and 40 ml aqueous hydrogenbromide (45%) and heated under reflux for 3 h. The solvents areevaporated and the residue is suspended in hot water and filtered. Thetwo isomeric products are separated by preparative HPLC:

M1: HPLC t_(R)=4.95 min, MS-APCI⁺: (M+H)⁺=390, m.p.: 297-299° C.;

M4: HPLC t_(R)=5.25 min, MS-APCI⁺: (M+H)⁺=390, m.p.: 312-315° C.

Step 2.1: Mixture of2-(2-Hydroxy-phenyl)-6-methoxy-benzo[e][1,3]oxazin-4-one with2-(2-hydroxy-5-methoxy-phenyl)benzo[e][1,3]oxazin-4-one

A mixture of 11 g 2-hydroxy-5-methoxy-benzoic acid, 7.5 g2-hydroxy-benzamide and 0.5 ml pyridine in 50 ml xylene is heated underreflux. 8.8 ml of thionylchloride is added over a period of 1 h. Themixture is kept under reflux for another hour, cooled to 40° C. and 100ml ethanol are added. After cooling to 5° C. the precipitated product isfiltered off and washed with cold ethanol; HPLC t_(R)=5.23 & 5.41 min,MS-APCI⁺: (M+H)⁺=270.

Step 2.2: Mixture of4-[3-(2-Hydroxy-5-methoxy-phenyl)-5-(2-hydroxy-phenyl)-[1,2,4]triazol-1-yl]-benzoicAcid with4-[5-(2-Hydroxy-5-methoxy-phenyl)-3-(2-hydroxy-phenyl)-[1,2,4]triazol-1-yl]-benzoicAcid

A mixture of 7 g of2-(2-hydroxy-phenyl)₆-methoxy-benzo[e][1,3]oxazin-4-one with2-(2-hydroxy-5-methoxy-phenyl)-benzo[e][1,3]oxazin-4-one and 4 g4-hydrazino-benzoic acid in 70 ml of ethanol are heated to reflux for 1h. The mixture is evaporated to dryness; HPLC t_(R)=5.56 & 5.67 min,MS-APCI⁺: (M+H)⁺=404.

1. A compound of the formula I

wherein one of the radicals R₁, R₂, R₃ and R₄ is hydroxy and theremaining radicals are each independently of the others hydrogen orhydroxy; or a salt thereof.
 2. A compound of the formula I according toclaim 1, which is4-[3-(2,3-dihydroxy-phenyl)-5-(2-hydroxy-phenyl)-[1,2,4]triazol-1-yl]-benzoicacid, or a salt thereof.
 3. A compound of the formula I according toclaim 1, which is4-[3-(2,5-dihydroxy-phenyl)-5-(2-hydroxy-phenyl)-[1,2,4]triazol-1-yl]-benzoicacid, or a salt thereof.
 4. A compound of the formula I according toclaim 1, which is4-[5-(2,5-dihydroxy-phenyl)-3-(2-hydroxy-phenyl)-[1,2,4]triazol-1-yl]-benzoicacid, or a salt thereof.
 5. A compound of the formula I, or apharmaceutically acceptable salt thereof, according to claim 1 for usein the treatment of diseases which cause an excess of metal in the humanor animal body or are caused by it.
 6. A compound of the formula I, or apharmaceutically acceptable salt thereof, according to claim 1 for usein the treatment of diseases which cause an excess of iron in the humanor animal body or are caused by it.
 7. A pharmaceutical preparationcomprising at least one compound of the formula I, or a pharmaceuticallyacceptable salt thereof, according to claim 1 together with at least onepharmaceutically acceptable carrier.
 8. Use of a compound of the formulaI, or a pharmaceutically acceptable salt thereof, according to claim 1for the production of a pharmaceutical preparation for the treatment ofan excess of metal in the human or animal body.
 9. Use of a compound ofthe formula I, or a pharmaceutically acceptable salt thereof, accordingto claim 1 for the production of a pharmaceutical preparation for thetreatment of an excess of iron in the human or animal body.